Project Summary The goal of this project is to determine the mechanisms underlying the molecular and cellular endocrinology of parathyroid hormone receptor (PTHR) signaling as it relates to phosphate and vitamin D balance. PTHR is uniquely expressed on both apical and basolateral membranes of the polarized cells that form renal proximal tubules. The biological consequences of bilateral PTHR expression or its origin are unknown. The premise of the proposal is that characterizing asymmetric PTHR actions will fill a major gap in our understanding of PTHR signaling and function and reconcile conflicting views of PTH action. Pilot studies show that PTHR signals from both basolateral and apical membranes but only basolateral PTHR activation induces transcription of the 1?-vitamin D hydroxylase (CYP27B1). Apical PTHR signaling primarily inhibits phosphate transport. The molecular and cellular mechanisms regulating PTHR actions are incompletely defined. The PTHR interacts at apical membranes with the PDZ scaffolding protein NHERF1, which tethers the receptor and regulates G-protein signaling and function. Absence of NHERF1 or its downregulation causes relocation of PTHR to basolateral membranes with an increased 1,25[OH]2D in mice and humans. Preliminary data show that Scribble, a basolateral PDZ protein, exerts a reciprocal effect, where downregulation causes accumulation of PTHR at apical membranes. We hypothesize that the polarized PTHR arrangement arises from the segregated location of NHERF1 and Scribble. We advance a research program to uncover new aspects of PTHR signaling in polarized kidney cells and test the central hypothesis that polarized PTHR expression is driven by the asymmetric location of the PDZ adaptor proteins, Scribble and NHERF, which in turn underlies the signaling bias of apical and basolateral PTHR actions on vitamin D and phosphate homeostasis. We propose three closely linked aims to evaluate this idea. The first two aims of address the hypothesis that basolateral PTHR activation preferentially stimulates the 1?-vitamin D hydroxylase, whereas apical PTHR signaling primarily blocks Pi transport. Aim 1 will determine the role of Scribble and NHERF in the generation of PTHR polarity and its asymmetric signaling in human kidney proximal tubule epithelial cells. Aim 2 uses live-cell FRET microscopy and other state-of-the art fluorescence techniques to characterize basolateral and apical membrane signaling properties of PTHR and the effect of NHERF1 and Scribble on biased G protein signaling. Aim 3 will delineate the in vivo actions of polarized proximal tubule PTHR by testing the role of Scribble on PTHR-dependent vitamin D and phosphate metabolism using a novel, conditional proximal tubule Scribble knockout mouse model that we generated. The outcomes will frame innovative therapeutic approaches targeting disorders of mineral metabolism.